The isolation of DNA is an important step in many biochemical and diagnostic procedures. For example, the separation of nucleic acids from the complex mixtures in which they are often found is frequently necessary before other studies and procedures, e.g. detection, cloning, sequencing, amplification, hybridization, and so on, can be undertaken. The presence of large amounts of cellular or other contaminating material, e.g. proteins or carbohydrates, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. In addition, DNA may contaminate RNA preparations and vice-versa. Thus, improved methods for the isolation of DNA from complex mixtures, such as blood, cells, and tissues, are demanded, not only from the preparative point of view, but also in the many methods in use today which rely on the identification of DNA diagnosis of microbial infections, forensic science, tissue and blood typing, and detection of genetic variations.
Nucleic acid isolation methods generally require an initial nucleic acid isolation step, to separate the nucleic acid from materials, e.g. protein, which may interfere in the hybridization and amplification techniques which are used. A range of methods are known for the isolation of nucleic acids, but generally, these rely on a complex series of extraction and washing steps and are time consuming and laborious to perform. Classical methods for the isolation of DNA from complex starting materials, such as blood, blood products, tissues, or other biological materials, involve lysis of the biological material by a detergent or chaotrope, possibly in the presence of protein degrading enzymes, followed by extraction options such as solid phase extraction (such as silica spin columns) or phenol extraction followed with ethanol precipitation.
Recently, paramagnetic beads based technology has been used for DNA isolation due to the popularity of robotic liquid handling instruments. Most magnetic beads based methods rely on lysing the sample with a detergent or chaotrope followed by binding DNA with magnetic beads. Those procedures are normally time consuming and complicated. Isolated DNA obtained from those methods always has potential contamination issues from proteins, RNA and other substances from the sample. Contamination with those substances can interfere with the downstream applications leading to irreproducible or false results. For blood DNA isolation, hemoglobin and RNA contamination commonly result from current magnetic beads based methods. Also, the volume of blood sample is always a limiting factor for blood DNA isolation due to the limited handling volume of the instruments. The volume limitation of the starting blood sample volume has become a very important issue for isolating DNA from blood using automated processes.
Improvements in methods for isolating nucleic acids are, thus, continually being sought, and more recently, other methods have been proposed which rely upon the use of a solid phase. U.S. Pat. No. 5,234,809 for example, describes a method where nucleic acids are bound to a solid phase in the form of silica particles, in the presence of a chaotropic agent such as a guanidinium salt, and thereby separated from the remainder of the sample. WO 91/12079 describes a method whereby nucleic acids are trapped on the surface of a solid phase by precipitation, in which alcohols and salts are generally used as precipitants.
U.S. Pat. No. 6,617,105 described a method that uses solid phase particles to specifically or non-specifically bind cells from a sample, the collected cells are then lysed and DNA is bound to the same solid phase particles and separated. However, this method still has to use extra measures to remove contaminates such as RNA and proteins.
While such methods speed up the nucleic acid separation process, a need still exists for improved methods which are quick and simple to perform, which enable good yields to be obtained without significant losses, and in particular which are readily amenable to isolating nucleic acids from cells in mixtures or environments where they may be present at low concentrations, as a preparative first step in isolating nucleic acids from target cells in nucleic acid based cell detection procedures.